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. 2000 Feb 1;97(3):1107–1112. doi: 10.1073/pnas.97.3.1107

Figure 5.

Figure 5

COPII proteins and nucleotides are sufficient to package Pho84p into COPII vesicles from wild-type membranes. Permeabilized spheroplasts prepared from wild-type cells expressing Pho84p-HA (EY0667) were used in vesicle budding reactions in vitro. Reactions contained: nucleotides only (ATP and GTP), COPII + Sar1p (purified COPII proteins supplemented with nucleotides and Sar1p), and COPII − Sar1p (COPII proteins with nucleotides but without Sar1p). Pho84p-HA was immunoprecipitated from 10% of the total reactions, and the vesicle fractions were derived from 60% of the total reactions. A second immunoprecipitation was then performed to detect the amount of Vph1p in the total reactions and vesicle fractions. The immunoprecipitated proteins were resolved separately on 8% SDS/PAGE and quantified with a PhosphorImager. The percent of Pho84p-HA or Vph1p in the vesicle fraction compared with the corresponding total reaction (histogram) was calculated from the amount of radiolabeled Pho84p-HA or Vph1p (Lower), respectively. T, Total reaction. V, Vesicle fraction. Data are reported as the mean values of three independent experiments, and the error bars indicate the standard deviation.